Background and purpose: Castration-resistant prostate cancer (CRPC) has been a major cause of tumor-associated death among men worldwide. The discovery of novel therapeutic approaches for CRPC remains imperative. Atractylenolide I (ATR-I), a prominent bioactive component from Atractylodes macrocephala, exhibits powerful anticancer potentials in various malignancies. Nevertheless, the ATR-I’s activity on CRPC and its underlying mechanism has not been reported. Experimental approach: An enzalutamide-resistant (EnzR) cell line was successfully constructed. The pharmacological effects of ATR-I were assessed in vitro and in vivo. The changes in the gene expression profiles after ATR-I treatment were analyzed using RNA sequencing (RNA-seq). Key results: ATR-I suppressed the proliferative and migratory abilities of CRPC cells, while triggering cell cycle arrest and apoptosis. In constructed EnzR cells, ATR-I also inhibited their proliferation and promoted apoptosis. Intriguingly, a combination of ATR-I with enzalutamide synergistically induced more apoptosis of EnzR cells. Mechanistically, RNA-seq results identified kinesin family member 15 (KIF15) as a potential target of ATR-I. KIF15 was up-regulated in prostate cancer (PCa), and its higher level was associated with poorer clinical outcomes. Further investigation showed that ATR-I inhibited the expression of KIF15 mRNA and protein, thus mediating ubiquitin-proteasomal degradation of AR/AR-V7. Finally, our in vivo experiment verified that ATR-I alone or in combination with enzalutamide retarded the growth of EnzR xenograft tumors. Conclusion and implications: This study elucidated the antitumor mechanism of ATR-I against CRPC. Notably, ATR-I may be a promising therapeutic drug for CPRC patients and enhance the response to enzalutamide in EnzR patients.